An Improved Method for the Crystallization of Trypsin

The yields of crystalline trypsin prepared by the original method (1) are generally far below the values to be expected if all of the trypsinogen were changed into trypsin. The specific activity of the enzyme also tends to be low. This is due to a second reaction (2) which occurs simultaneously with the transformation of trypsinogen into t iypsin--part of the trypsinogen is transformed into an "inert protein" which cannot be changed into trypsin by any known means. The formation of "inert protein" can be completely prevented (3), however, by allowing the autocatalytic conversion of trypsinogen to trypsin to proceed in the presence of calcium ions. Under these conditions, the trypsinogen is converted quantitative]y into trypsin which can then be easily crystallized. Both the yield and quality of the crystalline trypsin are greatly improved. The new method for the preparation of crystalline trypsin is as follows:-1. Conversion of Trypsinogen into Trypsin in the Presence of Calcium Chloride.--Dissolve 30 gin. dried crystalline trypsinogen 1 (or 50 gm. semidry filter cake) in 200 ml. 0.005 M hydrochloric acid. Add the solution to a previously prepared ice-cold mixture of 100 ml. 1 ~ calcium chloride, 250 ml. 0.4 M 2 borate buffer, pH 8.0, and 400 ml. distilled water. Adjust the volume of the mixture with water to 1000 ml., and leave in cold room at about 5°C. for 18 to 24 hours. 2. Removal of Calcium Ions.--Add 2 gm. standard super cel (Johns-Manville Corporation) and filter in cold room with suction through soft paper (Eaton-Dikeman No. 303). Reject filter cake. Adjust filtrate to pH 3.0 (tested with methyl orange on spot plate) with 5 N sulturic acid (about 4 rnl.). Add solid ammonium sulfate (242 gm. per 1000 ml.) to 0.4 saturation and leave in cold room for 2 days. A heavy sediment of CaSO4 crystals is formed. Filter with suction through soft paper (Whatman No. 3). Reject precipitate. 3. Precipitation of Amorphous Trypsin.--Add solid ammonium sulfate to